During FY2009, this laboratory has expanded structure-function studies on HIV Env-receptor interactions involved in virus entry, and on the development of HIV based on molecules involved in entry. 1) Based on our extension of a functional complementation system to quantitate cell fusion by mixed trimers containing Env protomers with different functional defects (Salzwedel and Berger, Retrovirology, 2009), we are testing the mechanism whereby the V1V2 loops mask conserved epitopes on V3. We have produced constructs with appropriate combinations of loop deletions and epitope mutations. Experiments are underway using both the cell fusion assay and HIV pseudotype infection assays to test whether epitope masking occurs by a "cis" or "trans" (within or between protomers, respectively). We are also using this complementation assay to study recently described neutralizing that appear to have epitopes consisting of determinants in both V2 and V3;constructs have been generated to test whether these epitopes are formed by "cis" or "trans" associations of these loop regions. 2) In collaborative studies of the gp120-CD4 interaction, we are exploring whether distinct regions of each protein are involved in high affinity bindiing versus "induction" of the coreceptor binding site. Appropriate mutant constructs have been designed for production of the corresponding proteins, in both membrane-associated and soluble forms. 3) sCD4-17b. This recombinant bifunctional protein was found to potently neutralize 100% of the nearly 4 dozen HIV-1 isolates tested (clades A, B, C, D, F, and recombinants AE and AG), with IC50 values ranging from <1 to 9 microgm/ml (Lagenaur et al., submitted). The breadth was considerably greater than any of the known broadly neutralizing antibodies. Moreover, sCD4-17b was found to be active not only on live virus particles generated from a cell line, but also the corresponding isogenic particles derived after a single passage through PBMC;by contrast, the broadly neutralizing monoclonal antibodies were considerably less sensitive to the PBMC-passaged virus, despite the absence of any sequence changes in Env (as shown by others). Based on this favorable neutralization profile, sCD4-17b was selected for further evaluation by the Alliance for Microbicide Development and the NIAID Microbicide Evaluation Group, as a candidate microbicide to inhibit HIV sexual transmission. Collaborations are continuing for high levell production of sCD4-17b in plant systems, to make this approach economically viable. 4) Anti-HIV immunotoxins for depletion of infected cell reservoirs persisting under HAART. We are continuing to formulate a pre-IND for a phase 1 clinical trial of 3B3-PE38. We have also initiated collaborative studies to test this concept in nonhuman primates. We have initiated collaborative studies to test immunotoxin complementation of antiretroviral therapy in SIV-infected macaques, for which models of viral peristence similar to that observed in HIV infection are being developed. Because our most favorable immunotoxin, 3B3-PE38, is not active against SIV Env, these proof-of-concept studies will be performed with our earlier agent sCD4-PE40. Promising efficacy results would greatly support clinical testing of 3B3-PE38.